Cryptococcus neoformans is a major pathogen in immunocompetent as well as immunocompromised patients including those with AIDS in both the developed as well as the developing world. Our long-term objective is to test the hypothesis that molecular regulators of the virulence factor laccase affect the virulence of Cryptococcus neoformans. The specific hypothesis behind the present proposal is that a virulence associated DEAD-box protein, Vad1, identified by insertional mutagenesis, is an important regulator of laccase and virulence in C. neoformans. This is based on the following observations. First, deletion of VAD1 results in loss of virulence and accelerated clearance of C. neoformans from lung in mouse models. Second, differential display has shown that deletion of VAD1 results in altered transcription of a number of genes in addition to laccase. Finally, deletion of one of the genes showing VAD1-dependent transcription, PCK1, exhibited attenuated virulence in a mouse model in spite of retained laccase activity. In Specific Aim 1, we will assess for functional conservation of VAD1 within the RCK/p54 protein subfamily of DEXD/H box proteins to which Vad1 exhibits significant homology. We plan to test this by overexpression of VAD1 in an RCK/p54 mutant of yeast, testing the role of VAD1 in haploid fruiting and mating, comparing the cellular localization of the Vad1 protein to members of the RCK/p54 subfamily and performing gel filtration of cellular extracts of cells expressing affinity-tagged Vad1 constructs to identify and characterize possible Vad1 multi-protein complexes. In Specific Aim 2, we will further define the role of VAD1 in laccase transcription. This will test our hypothesis that VAD1 is required for optimal expression of laccase. We will analyze VAD1 -mediated degradation of the transcriptional repressor, NOT1, and the role of NOT1 in suppression of laccase activity. In Specific Aim 3, we will analyze the host responses between wild-type and delta-vadl infections using an intratracheal model. Specifically, we will measure fungal cfu, leukocyte recruitment, cytokine production, antibody production and DTH response after infection with each strain. In Specific Aim 4, we will assess the role of 5 M4D7-dependent genes in cryptococcal virulence using an intratracheal and an intravenous mouse model. Completion of these specific aims will provide an integrated approach to understanding the role of VAD1 in the pathobiology of C. neoformans and may provide additional drug development targets for the treatment and prevention of cryptococcosis.